Project Summary The estrogen receptor-? (ER) and androgen receptor (AR) are the primary sex steroid hormone receptors in females and males, respectively. They drive reproduction-related gene expression programs by recruiting a series of coactivators (CoA) that form large, dynamic protein complexes at gene promoters and enhancers. While we know a great deal about these sex steroid hormone receptors at a functional and structural level, much less is known about the CoAs that interact with them at gene promoters and enhancers. Using advanced cryo-electron microscopy (cryo-EM) approaches, we were able to provide the first 3D understanding of ER engaged with CoA complexes at a gene enhancer. In this proposal, we plan to expand upon these findings by resolving the structure of receptor-CoA complexes on chromatin and with a more complete repertoire of proteins that comprise transcriptional supercomplexes. Also, in preliminary studies, we have made new breakthrough progress toward resolving an androgen receptor (AR)/SRC-2/DNA holocomplex. Importantly, this will generate a better understanding of NR/CoA holocomplexes to reveal new insights into the distinct roles for the N-terminal and C- terminal activation functions (AF-1 and AF-2) of both ER and AR. We will functionally interrogate the structures identified in our cryo-EM studies using innovative cell-free in vitro assay systems to understand how ER, AR, SRCs and co-CoAs distinctly regulate chromatin post-translational modifications, chromatin conformation and gene expression. Steroid receptor coactivators (SRCs) are the central scaffolding components of multi-protein NR/CoA/co-CoA complexes. New mechanistic insights into NR/CoA function must take into consideration the 3D structural conformation of the modular domains of NRs (DNA binding, AF-1 and AF-2 domains), CoAs and chromatin topology to generate conceptually novel insights into CoA biology and gene regulation by NRs. The studies proposed here should form a roadmap to therapy that can be used to develop new therapeutic drugs for reproductive disease states.